Vector for gene transfer into plant allowing optional deletion of marker gene

ABSTRACT

The present invention relates to a vector for introducing a desired gene into a plant, wherein a selectable marker gene introduced into a plant cell along with a desired gene is optionally removable from the DNA such as chromosome or the like where it exists and functions, then disappeared the function thereof after its expression, and the expression of the selectable marker gene and the disappearance of the function thereof are detectable by morphological change of the tissue derived from the plant cell into which the selectable marker gene is introduced. Also, the present invention constitutes a vector using a morphological abnormality induction gene as a selectable marker gene, while putting a removable DNA element under control of an inducible promoter, wherein the morphological abnormality induction gene is positioned such that it behaves integrally with the removable DNA element, and wherein a desired gene is positioned such that it does not behave integrally with the removable DNA element.

TECHNICAL FIELD

The present invention relates to a novel vector for introducing adesired gene into a plant using genetic engineering methods to obtain atransgenic plant.

BACKGROUND ART

Transformation of microorganisms and cultured cells using geneticengineering is currently applied to the production of physiologicallyactive substances useful as medicines and the like, and thus greatlycontributes to the industry. In the field of plant breeding, industrialapplication of genetic engineering lags behind because the life cyclesof plants are much longer than those of microorganisms and the like.However, since this technology enables a desired gene to be directlyintroduced into plants to be bred, it has the following advantagescompared to classical breeding which requires multiple crossing: (a) itis possible to introduce only a characteristic to be improved; (b) it ispossible to introduce characteristics of species other than plants (suchmicroorganisms and the like); and (c) it is possible to greatly shortenthe breeding period. Thus, genetic engineering methods for plantbreeding have been investigated vigorously.

Specifically, the production of transgenic plants requires the followingthree steps: (1) introducing the desired gene into the plant cell(including introduction of the same into the chromosomes, nucleus andthe like); (2) selecting plant tissue made only of cells into which thedesired gene has been introduced; and (3) regenerating a plant from theselected plant tissue. Furthermore, among these, in selecting the tissueinto which the desired gene has been introduced, a selectable markergene is generally used. In other words, generally, a selectable markergene is introduced into plant cells along with a desired gene, and acharacteristic feature shown by expression of the selectable marker genein the introduced cells, as well as a tissue derived from the cells, isused as an index for the introduction of the desired gene. Consequently,a selectable marker gene is introduced and expressed in addition to adesired gene in almost all cases of the plants so far transformed bymeans of genetic engineering methods.

However, with regard to the products of genes used as such selectablemarkers, their safety to the human body has been confirmed only on fewgenes. Accordingly, even if tomatoes or potatoes are produced byintroducing a useful character using a selectable marker gene, it willentail many obstacles, including a vague unrest in consumers, when theyare provided as edible products so long as the selectable marker gene isexpressed.

Furthermore, after selection of a gene-introduced tissue, expression ofa selectable marker gene will cause considerable obstacles even at thelevel of researchers studying on the plant breeding. That is, when atransgenic plant which has been produced by using a selectable markergene is again introduced by another gene, introduction of the genecannot be carried out using the same selectable marker gene. In otherwords, since the selectable marker gene has been already present in theplant, the selectable marker gene is always expressed in the plantwhether or not the new desired gene is introduced into the plant alongwith the selectable marker gene. Therefore, such a selectable markergene can no longer be used as an index of the introduction of the newdesired gene. Consequently, the number of times of repeated genetransfer into a certain plant is naturally restricted by the number ofdifferent selectable marker genes useful in the plant. However, kinds ofselectable marker genes so far available are not so many. Additionally,all of the selectable marker genes are not necessarily useful in theplant of the object.

For resolving the above-described problems and thereby efficientlyproducing a gene-introduced tissue or plant completely free from theinfluence of a selectable marker gene, the present inventors havealready developed a novel vector for introducing a desired gene intoplant cells (Japanese Patent Application No. H07-313432). This vectorcomprises a desired gene, a morphological abnormality induction gene asa selectable marker gene, and a removable DNA element, wherein themorphological abnormality induction gene is positioned such that itbehaves integrally with the removable DNA element, and wherein thedesired gene is positioned such that it does not behave integrally withthe removable DNA element. When a desired gene is introduced into aplant using this vector, the selectable marker gene is removable fromthe DNA where it exists and functions, then disappeared the functionthereof at a certain ratio after its expression through cultivation oftransgenic cells, and the expression of the selectable marker gene andthe disappearance of the function thereof can be detected bymorphological change of the tissue derived from the plant cell intowhich the selectable marker gene is introduced. That is, a tissuederived from the cell in which this selectable marker gene is expressedshows a certain abnormal morphology, and, when cells from which thefunction of the selectable marker gene is disappeared by the removalthereof (in other words, cells into which only the desired gene isintroduced) are generated from the tissue thereafter, a tissue havingnormal morphology is regenerated from the resulting cell. Accordingly,by using this vector, a plant tissue comprising cells into which only adesired gene is introduced, as well as its subsequent plant individual,can be produced by simply repeating culturing of the gene-introducedcells and selection of tissues obtained by the culturing visually.

However, the removal of the selectable marker gene could not be freelycontrolled even in this vector developed by the present inventors.Accordingly, if the ability of the removable DNA element is high, theselectable marker gene will be removed very quickly. For example, theselectable marker gene will be removed immediately after itsintroduction into plant cells along with a desired gene and before itsexpression. In this case, a tissue constituted by cells into which onlythe desired gene is introduced may be obtained; however, the selectablemarker gene-induced morphological changes of the gene-introduced tissuedo not occur and, as the result, such a tissue cannot be selected.

Additionally, if the removal of the selectable marker gene can be freelycontrolled, the generation of cells into which only a desired gene isintroduced and the generation of plant tissues derived from such cellscan be synchronized or appropriately controlled, and therefore, it willbe very convenient in actually producing a transgenic plant using such avector.

Consequently, an object of the present invention is to provide a vectorfor introducing a gene into a plant, which contains a selectable markergene, in which functions of the selectable marker gene introduced intoplant cells together with a desired gene can be optionally removableafter its expression by removing the selectable marker gene from DNA,such as chromosomal or the like, where it exists and functions and theexpression and disappearance of the function of the selectable markergene can be detected by the morphological changes of tissues derivedfrom the gene-introduced plant cells.

DISCLOSURE OF THE INVENTION

The object of the present invention can be accomplished by constructinga vector using a morphological abnormality induction gene as aselectable marker gene, while putting a removable DNA element undercontrol of an inducible promoter, wherein the morphological abnormalityinduction gene is positioned such that it behaves integrally with theremovable DNA element, and wherein a desired gene is positioned suchthat it does not behave integrally with the removable DNA element.

The present invention will be discussed below in detail.

As used therein, the morphological abnormality induction gene is a genethat induces into a tissue of a plant morphologically abnormaldifferentiation such as a dwarf, destruction of apical dominance, changein pigments, formation of a crown gall, formation of hairy roots, wavingof the leaves or the like. It is reported that various morphologicalabnormality induction genes, such as cytokinin synthesis genes (e.g.,ipt (isopentenyltransferase) gene (A. C. Smigocki, L. D. Owens, Proc.Natl. Acad. Sci. USA, 85:5131, 1988)), iaaM (tryptophan monooxygenase)gene (H. J. Klee et al., GENES & DEVELOPMENT, 1:86, 1987), gene 5 (H.Koerber et al., EMBO Journal, 10:3983, 1991), gene 6b (P. J. J. Hooyaaset al., Plant Mol. Biol., 11: 791, 1988), rol genes such as rolA to D(F. F. White et al., J. Bacteriol., 164:33, 1985) and the like, arepresent in bacteria of the genus Agrobacterium or the like that inducetumor or teratoma in various plants (that is, formation of adventitiousshoots or adventitious roots). Furthermore, an iaaL (indoleaceticacid-lysine synthetase) gene in Pseudomonas syringae subsp. savastanoi(A. Spena et al., Mol. Gen. Genet., 227:205, 1991), and homeo box genes,phytochrome genes and the like in various plants are reported.

Any of these genes can be used in the present invention. Among these,the ipt gene which induces destruction of apical dominance and the rolgenes which induces the formation of hairy roots, dwarf, waving of theleaves and the like of a plant regenerated from the hairy root arepreferable selectable marker genes in the present invention because theyinduce characteristic morphological abnormality among variousmorphological abnormality induction genes.

Furthermore, one can design a combination of these selectable markergenes, so that a specific structure, such as an adventitious shoot, anadventitious root or the like is redifferentiated in a specific plantinto which these selectable marker genes are introduced. In the presentinvention, such a combination of morphological abnormality inductiongenes can be used, according to the conditions of producing thetransgenic plant, such as the kind of a plant into which the genes areto be introduced.

As used herein, a removable DNA element is an element of a DNA sequencewhich itself is removable from the DNA wherein the DNA element existsand functions. In plants, a transposon present in a chromosome is knownas this element. The structure, activity and behavior of transposonshave been almost completely identified. For the transposon to function,two components are required in principle, an enzyme which is expressedfrom the gene present therein and which catalyzes the excision andtransposition of the transposon itself (transposase), and DNA bindingsequences which are present in the terminal region of the transposon andupon which the transposase acts. By these elements, the transposon isexcised from the chromosome in which it exists, and is then usuallytransposed to a new position in the DNA. However, at a certain ratio,the transposon also disappears without being transposed. The presentinvention makes use of such a transposition error of the transposon.

The transposon can be of one of two types, either an autonomoustransposon or a non-autonomous transposon. The autonomous transposonmaintains the two elements, the transposase and the DNA bindingsequence. In the autonomous transposon, the transposase is expressed andbinds to the DNA binding sequence for action, whereby the transposon isautonomously excised from the chromosome. The non-autonomous transposonretains the terminal DNA binding sequence to which the transposase isbound for action. In the non-autonomous transposon, the transposase geneundergoes mutation such that the transposase is not expressed; thus thetransposon cannot be excised from the chromosome autonomously. However,when transposase is supplied to the non-autonomous transposon from theautonomous transposon or from an independent transposase gene, thenon-autonomous transposon behaves similarly to the autonomoustransposon.

Accordingly, in the present invention, both the autonomous andnon-autonomous transposons can be used. For example, a non-autonomoustransposon can be used by inserting therein a morphological abnormalityinduction gene and a transposase gene which is obtained from anautonomous transposon or synthesized.

Examples of the autonomous transposons include Ac and Spm isolated frommaize (A. Gierl and H. Saedler, Plant Mol. Biol., 19:39, 1992). Ac canbe obtained by digesting wx-m7 locus in the chromosome of the maize withrestriction endonuclease Sau3A (U. Behrens et al., Mol. Gen. Genet.,194:346, 1984). This autonomous transposon is the most analyzed amongplant transposons. In fact, the DNA sequence has already been determined(M. Mueller-Neumann et al., Mol. Gen. Genet., 198:19, 1984). Also,examples of non-autonomous transposons include Ds and dspm obtained bydeleting the inner regions of Ac and Spm, respectively (H.-P. Döring andP. Starlinger, Ann. Rev. Genet., 20:175, 1986) and those isolated frommany plants, other than maize, such as snapdragon, morning glory and thelike (for example, Y. Inagaki et al., Plant Cell, 6:375, 1994). Whenthese transposons are introduced into chromosomes of exogenous plants,these transposons are also excised from a chromosome and transposed (forexample, B. Baker et al., Proc. Natl. Acad. Sci. USA, 83:4844, 1986).

Another removable DNA element, which is not present in plants, is anelement derived from a site-specific recombination system. Asite-specific recombination system consists of two elements, arecombination site (corresponding to the removable DNA element of thepresent invention) having a characteristic DNA sequence, and an enzyme(recombinase) that binds to the DNA sequence specifically and catalyzesthe recombination between these DNA sequences if two or more of thesequences exist. When the two DNA sequences are oriented in the samedirection at a given interval on the same DNA molecule, the region heldby these DNA sequences is excised from the DNA molecule, such as aplasmid, chromosome or the like. When the two DNA sequences are orientedin opposite directions on the same DNA molecule, the region held bythese DNA sequences is inverted. The present invention utilizes theformer excision. Both excision and inversion within the recombinationsite occur as a result of homologous recombination through thesite-specific recombination system, which is different from themechanism using the transposon. It is known that the recombinase gene isnot necessarily present in the same DNA molecule, in which therecombination site exist. The recombinase gene only needs to be presentin the same cell and expressed to excise or invert the region held bythe two DNA sequences (N. L. Craig, Annu. Rev. Genet., 22:77, 1988).

At present, site-specific recombination systems have been identified inmicroorganisms such as phage, bacterium (e.g., E. coli), yeast and thelike. Examples thereof include a Cre/lox system, a pSR1 system, a FLPsystem, a cer system, and a fim system (for example, N. L. Craig, Annu.Rev. Genet., 22:77, 1988). When the site-specific recombination systemseparated from these microorganisms with the use of a Cre/lox systemderived from P1 phage (International Laid-Open No. WO 93/01283) isintroduced into organisms (including plants) different from the organismfrom which this system had been derived, it behaves in the same way asin the original organism. The site-specific recombination system ofyeast (Zygosaccharomyces rouxii) (pSR1 system (H. Matsuzaki et al., J.Bacteriology, 172:610, 1990)) can also be used in accordance with thepresent invention. This pSR1 system also maintains its inherent functionin higher plants (H. Onouchi et al., Nucleic Acid Res., 19:6373, 1991).

According to the present invention, this removable DNA element is putunder control of an inducible promoter.

That is, inducible promoters are present in upstream of structural genesin all organisms ranging from prokaryotic organisms (for example,bacteria and the like) to eucaryotic organisms (for example, yeasts,fungi, higher plants, mammals and the like), and these elements controlexpression of a certain gene or a group of genes by functioning alone orin concert with one another. For example, they carry out on/off of thegene expression sometimes in response to the stage of differentiationand growth of each individual or occasionally depending on the heat,light, metals and the like environmental factors. According to thepresent invention, an inducible promoter having such functions is used,and a removable DNA element positioned in its downstream controlsexpression, or removability.

Examples of such inducible promoters so far known include those whichrespond to chemical substances, such as glutathione-S-transferase Isystem gene promoter (Unexamined Published Japanese Patent ApplicationNo. H05-268965), glutathione-S-transferase II system (GST-II) genepromoter (International Laid-Open No. WO 93/01294), Tet repressor fusiontype cauliflower mosaic virus 35S promoter (C. Gatz et al., Mol. Gen.Genet., 227:229, 1991), Lac operator/repressor system promoter (R. J.Wilde et al., The EMBO Journal, 11:1251, 1992), alcR/alcA systempromoter (International Laid-Open WO 94/03619), glucocorticoid systempromoter (Takushi Aoyama, Protein, Nucleic acid and Enzyme, 41:2559,1996) and the like, those which respond to heat, such as hsp80 promoter(Unexamined Published Japanese Patent Application No. H05-276951) andthe like and those which respond to light, such as ribulose-bisphosphatecarboxylase small subunit gene (rbcS) promoter (R. Fluhr et al., Proc.Natl. Acad. Sci. USA, 83:2358, 1986), fructose-1,6-bisphosphatase genepromoter (Japanese Domestic Re-publication of PCT InternationalPublication for Patent Application No. H07-501921), light-harvestingchlorophyll a/b binding protein gene promoter (Unexamined PublishedJapanese Patent Application No. H05-89) and the like.

Among these promoters, the rbcS promoter has been studied mostprogressively as an inducible promoter in higher plants and analyzedmore in detail by Chua et al. (for example, see Matsuoka, Plant CellTechnology Supplement, 3:552, 1991). Because of this reason, thispromoter was used in Example 1 of the present invention, but themechanism of this factor in regulating gene expression in response tolight has not been established yet. On the other hand, promoters whichrespond to chemical substances, typically including the GST-II genepromoter, can control induction of gene expression relatively freely inresponse to the amount of chemical substances, so that they have anadvantage in practical use in comparison with heat- or light-respondingpromoters which have to control gene expression by controlling heat orlight.

In the present invention, the morphological abnormality induction genemay be inserted into a position where this gene is excised along withthe removable DNA element. For instance, when the transposon is used asthe removable DNA element, the morphological abnormality induction genecan be inserted into a position which does not influence the excision ofthe transposon and which is upstream of the promoter region of thetransposase gene but downstream of the terminal region to which thetransposase binds. When the pSR1 system is used, the morphologicalabnormality induction gene can be inserted into any position within theregion held by the recombination sites which does not inhibit theexpression of the recombinase.

The vector of the present invention can be used in any plants into whichthe gene can be introduced by genetic engineering methods. The desiredgene in accordance with the present invention can be any gene by whichagriculturally excellent characteristics can be imparted and any genewhich allows for studies of gene expression mechanisms and the like,though agriculturally excellent characteristics are not necessarilyimparted.

With regard to a promoter and a terminator for the desired gene, theycan be used without any restrictions so long as they function in plants.Examples of promoter include the 35S promoter of a cauliflower mosaicvirus (J. T. Odell et al., Nature (London), 313:810, 1985), the promoterof a nopaline synthetase (W. H. R. Langridge et al., Plant Cell Rep.,4:355, 1985), and the like. Examples of terminator include thepolyadenylation signal of a nopaline synthetase (A. Depicker et al., J.Mol. Appl. Gen., 1:561, 1982), the polyadenylation signal of an octopinesynthetase (J. Gielen et al., EMBO J., 3:835, 1984), and the like.

Furthermore, the gene, that is, DNA, of the present invention can beobtained by cloning cDNA or genomic DNA, or by chemical synthesis if itssequence is known.

The vector of the present invention can be indirectly introduced intothe plant cell through viruses or bacteria with which plants areinfected (I. Potrykus, Annu. Rev. Plant Physiol. Plant Mol. Biol.,42:205, 1991). Examples of viruses include cauliflower mosaic virus,geminivirus, tobacco mosaic virus, brome mosaic virus, and the like.Examples of bacteria include Agrobacterium tumefaciens (hereinafterreferred to as A. tumefaciens), Agrobacterium rhizogenes, and the like.Dicotyledonous plants are generally known to be infected with thebacteria of the genus Agrobacterium. Recently, the introduction of genesinto the monocotyledonous plants by the infection of these plants withthem has also been reported (for example, International Laid-Open No. WO94/00977).

The vector of the present invention can be directly introduced into theplant cell by physical and chemical methods such as a microinjection, anelectroporation, a polyethylene glycol method, a fusion method, ahigh-speed ballistic penetration, and the like (I. Potrykus, Annu. Rev.Plant Physiol. Plant Mol. Biol., 42:205, 1991). Since the generalindirect introduction method using the genus Agrobacterium cannot beapplied to many of the monocotyledonous plants and the dicotyledonousplants which are resistant to infection with Agrobacterium, theabove-mentioned direct introduction methods are effective for theseplants.

[Effects]

In the present invention, the morphological abnormality induction geneis expressed to make the cell physiological abnormal. Physiologicalabnormalities include the production of plant growth hormone in a plantcell, with the result that the proliferation and differentiation of thecell containing the morphological abnormality induction gene areconfused to induce various morphological abnormalities. For example, anaggregate of disordered shoots with the apical dominance destroyed(extreme shooty phenotype), hairy roots or the like, can be derived froma cell into which such a morphological abnormality induction gene isintroduced. This phenotype is formed by abnormal proliferation anddifferentiation of the above-mentioned cell. Thus, this morphologicallyabnormal tissue is made up only of the cell containing this gene.Accordingly, if the vector is constructed using this gene as theselectable marker gene together with the desired gene and is introducedinto the plant cell and the cell is cultured, the tissue made up only ofthe cell into which the selectable marker gene and the desired gene havebeen introduced can be selected by merely visually selecting themorphologically abnormal tissue derived from the plant cell.

Furthermore, according to the present invention, the morphologicalabnormality induction gene is used by incorporating it into a positionwhere it behaves integrally with the removable DNA element which is putunder control of an inducible promoter. When a gene is introduced into aplant by using a vector having such a construction, the removable DNAelement can be expressed by artificially applying an appropriatestimulus such as heat, light, a chemical substance or the like dependingon the used inducible promoter to the plant cells after the genetransfer, so that the morphological abnormality induction gene as aselectable marker gene disappears its function by removing along withthis removable DNA element, at a certain ratio from the DNA moleculewhere they were once introduced and functioned, while the desired genewhich does not behave integrally with it remains on the same DNAmolecule and maintains its function, thus, in other words, cells inwhich only the desired gene is introduced can be obtained.

Moreover, since the disappearance of the function of this selectablemarker gene, namely the disappearance of the function of themorphological abnormality induction gene, can be visually detected asmorphological change of the gene introduced-tissue in the same manner asin the introduction of the gene, the tissue made up only of the cells inwhich the function of the selectable marker gene has been disappeared,in other words, the tissue made up only of the cells in which only thedesired gene is introduced, can be selected surely and easily. That is,in order to obtain the tissue made up only of such cells, the cellsafter the gene introduction are cultured, the tissue showingmorphological abnormality, such as shooty phenotype, hairy roots or thelike, caused by the expression of the morphological abnormalityinduction gene is visually selected, and the selected tissue isseparated. Then, if appropriate stimulus is optionally applied theretoaccording to the used inducible promoter during culturing of theseparated tissue, a tissue showing morphological normality is obtainedthis time from the tissue showing morphological abnormality, and canalso be visually selected.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows steps until preparation of pNPI201 in the pNPI206construction scheme.

FIG. 2 shows steps for the preparation of pNPI201 to pNPI203 in thepNPI206 construction scheme.

FIG. 3 shows steps for the preparation of pNPI203 to pNPI204 in thepNPI206 construction scheme.

FIG. 4 shows steps for the preparation of pNPI204 to pNPI206 in thepNPI206 construction scheme.

FIG. 5 is a restriction enzyme map of the T-DNA region in the pNPI206structure.

FIG. 6 shows construction scheme of pNPI125.

FIG. 7 shows construction scheme of pNPI128.

FIG. 8 shows steps until preparation of pIPT2 in the pNPI123construction scheme.

FIG. 9 shows steps for the preparation of pIPT2 to pIPT3 in the pNPI123construction scheme.

FIG. 10 shows steps for the preparation of pIPT3 to pIPT4 in the pNPI123construction scheme.

FIG. 11 shows steps for the preparation of pIPT4 to pNPI123 in thepNPI123 construction scheme.

FIG. 12 shows steps for the preparation of pNPI200 and pG1E7 to pNPI301in the pNPI303 construction scheme.

FIG. 13 shows steps for the preparation of pNPI123 and pNPI128 to pIPT8in the pNPI303 construction scheme.

FIG. 14 shows steps for the preparation of pNPI301 and pIPT8 to pNPI302in the pNPI303 construction scheme.

FIG. 15 shows steps for the preparation of pNPI302 to pNPI303 in thepNPI303 construction scheme.

BEST MODE OF CARRYING OUT THE INVENTION EXAMPLES

In the following Examples, the experiments were conducted according tothe instructions of Molecular Cloning, 2nd edition (Sambrook et al.eds., Cold Spring Harbor Laboratory Press, New York, 1989) or through amanufacturer unless otherwise instructed.

Example 1

1. Construction of Vector

The recombinase gene (hereinafter referred to as “R gene”) of a yeastsite-specific recombination system (pSR1 system) and the nopalinesynthetase polyadenylation signal linked thereto were cut out withrestriction endonucleases XbaI and EcoRI from the plasmid pNPI125described in Japanese Patent Application No. H07-313432 and insertedbetween the XbaI-EcoRI restriction endonuclease site of pHSG398(purchased from Takara Shuzo Co., Ltd.) to obtain a plasmid pNPI200.

On the other hand, a plasmid containing the ribulose-bisphosphatecarboxylase small subunit gene promoter (rbcS-3B) (obtained from Dr.Mamoru Sugita of Nagoya University) was digested with a restrictionendonuclease KpnI, and the cohesive termini thus produced by thedigestion were changed into blunt-ended termini with T4 polymerase I(large subunit). Then, 5′-phosphorylated SalI linker was insertedbetween the resulting blunt-ended termini to obtain a plasmid pRBCS, andthe rbcS-3B was cut out from the pRBCS with a restriction endonucleaseSalI and inserted into the SalI restriction endonuclease site of pNPI200to obtain a plasmid pNPI201.

Also, the rbcS-3B is a light-responding promoter derived from a tomato(Lycopersicon esculentum VFNTLA 1221). In addition to this promoter,tomatoes have five other similar promoters (rbcS-1, -2, -3, -3A and -3C)as inducible promoters of rbcS, and their expression modes have beenanalyzed by Sugita et al. (M. Sugita et al., Proc. Natl. Acad. Sci. USA,84:7104, 1987).

Next, the plasmid pNPI201 was digested with restriction endonucleasesPstI and BamHI, and the cohesive termini thus produced by the digestionwere changed into blunt-ended termini with T4 polymerase I (largesubunit) and then ligated to obtain a plasmid pNPI202. Using restrictionendonucleases EcoRI and HindIII, a fragment containing rbcS-3B, R geneand nopaline synthase polyadenylation signal was cut out from theplasmid pNPI202 and inserted between the EcoRI-HindIII restrictionendonuclease site of pUC119 (purchased from Takara Shuzo Co., Ltd.) toobtain a plasmid pNPI203, and the fragment containing rbcS-3B, R geneand nopaline synthase polyadenylation signal was again cut out from theplasmid pNPI203 with restriction endonucleases EcoRI and HindIII andinserted between the EcoRI-HindIII restriction endonuclease site of theplasmid pNPI128 described in Japanese Patent Application No. H07-313432to obtain a plasmid pNPI204.

Thereafter, into the HindIII restriction endonuclease site of the thusobtained plasmid pNPI204 was inserted a fragment containing cauliflowermosaic virus 35S promoter (CaMV35S promoter) and ipt gene connectedthereto, which had been cut out from the plasmid pNPI123 also describedin Japanese Patent Application No. H07-313432 using the restrictionendonuclease HindIII, to obtain a plasmid pNPI205.

The desired vector could be obtained by cutting out fragment containingthe ipt gene linked to the CaMV35S promoter, the R gene and nopalinesynthase polyadenylation signal linked to the rbcS-3B and therecombination sequence Rs of yeast site-specific recombination systemlocated on both termini thereof from the plasmid pNPI205 with arestriction endonuclease PstI and inserting it into the SseI restrictionendonuclease site of a vector plasmid pBI121 for use in gene transferinto plants (purchased from TOYOBO CO., LTD.), and the thus obtaineddesired vector was named plasmid pNPI206. When a plant is infected withA. tumefaciens containing this plasmid, a T-DNA region which existsbetween an RB site and LB site of the plasmid, in this case, a region ofabout 12.5 kb from the nptII gene (neomycin phosphorylation enzyme gene)to GUS gene (β-glucronidase gene), is integrated into the plantchromosome.

Also, the plasmid pNPI206 was introduced into Escherichia coli JM109strain, and the resulting strain was applied to international depositionas E. coli JM109 (pNPI206) [National Institute of Bioscience and HumanTechnology, Agency of Industrial Science and Technology, the Ministry ofInternational Trade and Industry (Higashi 1-1-3, Tsukuba-shi, Ibaraki,Japan, 305), international accession number FERM BP-5518, originaldeposition under Budapest Treaty on Apr. 24, 1996].

The construction scheme of pNPI206 is shown in FIGS. 1 to 4 and therestriction endonuclease map of its T-DNA region is shown in FIG. 5.Also, the construction scheme of pNPI125 is shown in FIG. 6, theconstruction scheme of pNPI128 is shown in FIG. 7, and the constructionscheme of pNPI123 is shown in FIGS. 8 to 11. In these drawings, “35S-P”represents cauliflower mosaic virus 35S promoter, “NOS-P” representsnopaline synthase promoter, “T” represents nopaline synthasepolyadenylation signal, “encircled T” represents polyadenylation signalof ipt gene itself, and “triangle of half-tone dot meshing” representsrecombination sequence Rs and its sequencing direction.

As apparent from FIG. 5, this plasmid contains the ipt gene as aselectable marker gene and the nptII gene and GUS gene as models of thedesired gene in the T-DNA region, namely a region to be integrated intothe plant chromosome. This ipt gene is a member of tumor-inducing genespossessed by the pathogenic A. tumefaciens, and its introduction intoplant cells induces over production of a plant hormone, cytokinin, anddifferentiation of the resulting cells is directed toward extreme shootyformation. Also, both of the nptII gene which contributes to kanamycinresistance and the GUS gene that produces a blue pigment in cellscontaining the gene by metabolizing a specific substrate are genesgenerally used in the analysis of gene expression in plants.

In addition, since a region between a pair of recombination sequenceRs's of a yeast site-specific recombination system (pSR1 system)functions as the removable DNA element in this plasmid, the ipt gene isinserted in such a form that it is sandwiched by the set of therecombination sequence Rs's having the same direction. At the same time,however, the R gene as a gene of an enzyme which catalyzes removing ofthe region between Rs's is connected to downstream of an induciblepromoter, namely the light-responding promoter rbcS-3B, so that the geneis not expressed by regulation of the promoter, or the removing betweenRs's does not occur, unless it is put under appropriate lightconditions.

II. Introduction of pNPI206 into Agrobacterium

A. tumefaciens strain LBA4404 (purchased from CLONTECH CO., LTD.) wasinoculated into 10 ml of YEB liquid culture medium (containing 5 g/l ofbeef extract, 1 g/l of yeast extract, 1 g/l of peptone, 5 g/l ofsucrose, and 2-mM MgSO₄, pH of 7.2 at 22° C. (the pH at 22° C. isapplied to the following unless otherwise instructed)), and was culturedat 28° C. until OD₆₃₀ was within the range of 0.4 to 0.6. Then, theculture was centrifuged at 6,900×g for 10 minutes at 4° C. to collectthe cells. The cells were suspended in 20 ml of 10-mM Tris-HCl (pH 8.0),and the suspension was recentrifuged at 6,900×g for 10 minutes at 4° C.Subsequently, the collected cells were resuspended in 200 μl of YEBliquid culture medium, and this suspension was used as a cell suspensionfor plasmid introduction.

Introduction of pNPI206 into Agrobacterium was carried out by mixing 200μl of the cell suspension for plasmid introduction with 6 μg of thepNPI206 obtained in the above-described step I in a 15 ml capacity tube(manufactured by Falcon), cooling the mixture by soaking the tube for 5minutes in ethanol which had been cooled in advance for 30 to 40 minutesin liquid nitrogen, putting the tube for 25 minutes in a water bath of29° C., adding 750 μl of YEB liquid medium to the tube and thenculturing the cells in the tube at 29° C. for 1 hour on a shaker.

III. Introduction of pNPI206 from Agrobacterium into Tobacco, Culturingof pNPI206-introduced Tobacco Cells and Morphology of the ObtainedTissue

Matured leaves of a tobacco (Nicotiana tabacum cv. SR1) grown in agreenhouse were dipped in a 1 v/v% sodium hypochlorite aqueous solutionfor sterilization, and washed three times with sterile water. Then, themidrib of the leaf was removed to form leaf discs of approximately 8 mmsquare. The thus-obtained leaf discs were then dipped for approximately1 minute in a cell suspension of A. tumefaciens strain LBA4404introduced pNPI206 in the above-described step II, and was infectedtherewith (which suspension was diluted with a sterilized water atOD₆₃₀=0.25 after the overnight culturing in YEB liquid culture medium).The infected leaf disc was put on a sterilized filter paper to removeany extra cell suspension. Then, it was laid on hormone-free MS agarculture medium (T. Murashige and F. Skoog, Physiol. Plant., 15:473, 1962(provided that 0.8 w/v% agar was added thereto)) containing 50 mg/l ofacetosyringone with the back of the leaf facing upward, and was culturedfor 3 days, at 25° C. in a dark place (hereinafter, culturingtemperature of a plant tissue was 25° C. unless otherwise instructed).Next, when this was transplanted into hormone-free MS agar culturemedium containing only 500 mg/l of carbenicillin and cultured under aquantity of light of approximately 7 to 10 μmol s⁻¹m⁻² whilesubculturing with the medium having the same components, 225 extremeshooty phenotype lines were obtained after 3 months of the infection, sothat 126 lines among them were divided into two groups and culturing ofeach group was continued under a quantity of light of approximately 7 to10 μmol s⁻¹m⁻² or of approximately 70 μmol s⁻¹ m⁻² using the mediumhaving the same components.

As the results, shoots having normal morphology visually (hereinafterreferred to as a “normal individual”) were generated from 43 extremeshooty phenotype lines after about 6 months of the infection in thelines cultured under a quantity of light of approximately 70 μmols⁻¹m⁻², while only 12 extreme shooty phenotype lines generated normalindividuals after the same period of time in the lines cultured under aquantity of light of approximately 7 to 10 μmol s⁻¹m⁻².

The results are shown in Table 1.

TABLE 1 Light conditions and generation ratio of normal individuals incultured tobacco tissue transformed with pNPI206 The number of extremeshooty The number of lines Generation ratio of Light phenotype linesused for the which generated normal individuals conditions*¹ examinationof light conditions normal individuals (%)*² L2 → L1 126 43 34.1 L2 → L2126 12 9.5 Three months of culturing under L2 condition and then threemonths of culturing under L1 or L2 condition *¹Light conditions L2; aquantity of light of approximately 7 to 10 μmol s⁻¹m⁻² L1; a quantity oflight of approximately 70 μmo1 s⁻¹ m⁻² *²(the number of lines whichgenerated normal individuals/the number of extreme shooty phenotypelines used for the examination of light conditions) × 100

As apparent from the results shown in the above table, when the culturedtobacco tissue transferred with the vector pNPI206 of the presentinvention is cultured under low light conditions and then cultured underhigh light conditions, the normal individual generation ratio becomesalmost 4 times higher than that of continued culturing under the lowlight conditions. Accordingly, it is shown that behavior of themorphological abnormality induction gene used as a selectable markergene is controlled by the light responding promoter rbcS-3B used in theregulation of the removable DNA element, in the gene-introduced tissueusing pNPI206, and that its removing is accelerated by shifting lightconditions of the gene-introduced tissue from low light quantity to highlight quantity.

Example 2

1. Construction of Vector

The plasmid pNPI200 obtained in Example 1 was digested with PstI, andthe cohesive termini thus produced by the digestion were changed intoblunt-ended termini with T4 polymerase I (large subunit). The GST-II 27kD subunit gene (GST-II-27) promoter (Japanese Domestic Re-publicationof PCT International Publication for Patent Applications No. H06-511385,lines 15 to 17 in the right-side lower column on page 7), which had beencut out from plasmid pG1E7 (obtained from Zeneca Limited) with arestriction endonuclease NdeI and also subjected to blunt-ending of itsresulting cohesive termini with T4 polymerase I (large subunit) wassubsequently inserted between the blunt-ended termini of pNPI200 toobtain a plasmid pNPI300. Next, a fragment containing the GST-II-27promoter, R gene and nopaline synthase polyadenylation signal was cutout from the thus obtained pNPI300 with a restriction endonuclease EcoRIand inserted into the EcoRI restriction endonuclease site of pUC18(purchased from Takara Shuzo Co., Ltd.) to obtain a plasmid pNPI301.

GST-II is an enzyme which exists in corn and the like and is one of theisozymes of GST that concerns in the herbicide detoxification. Also, theGST-II-27 promoter controls gene expression of the 27 kD subunit as oneof the GST-II subunits, and it is known that this promoter dramaticallyincreases the GST-II activity to improve resistance of corn and the likeagainst herbicides, by inducing expression of GST-II-27 in the presenceof a herbicide antidote, such as2,2,5-trimethyl-3-(dichloroacetyl)-1,3-oxazolidine or analogs thereof(Japanese Domestic Re-publication of PCT International Publication forPatent Applications No. H06-511385).

On the other hand, CaMV35S promoter and ipt gene linked thereto were cutout from the plasmid pNPI123 with a restriction endonuclease HindIII andinserted into the HindIII restriction endonuclease site of the pNPI128to obtain a plasmid pIPT8. Then, a fragment containing the GST-II-27promoter, R gene and nopaline synthase polyadenylation signal was cutout from the pNPI301 with a restriction endonuclease EcoRI and insertedinto the EcoRI restriction endonuclease site of the thus obtained pIPT8to obtain a plasmid pNPI302.

The desired vector can be obtained by cutting out a fragment containingthe ipt gene linked to the CaMV35S promoter, the R gene and nopalinesynthase polyadenylation signal linked to the GST-II-27 promoter and therecombination sequence Rs's of yeast site-specific recombination systemlocated on both termini thereof from the plasmid pNPI302 with arestriction endonuclease SseI and inserting the fragment into the SseIrestriction endonuclease site of the vector plasmid pBI121 forintroducing a gene into a plant, and the thus obtained desired vectorwas named plasmid pNPI303. That is, in this plasmid pNPI303, theGST-II-27 promoter was used to control the R gene instead of the rbcS-3Bused in pNPI206. Construction scheme of pNPI303 is shown in FIGS. 12 to15. Symbols used in these drawings are the same as those used in FIGS. 1to 11.

Furthermore, the plasmid pNPI303 was also introduced into Escherichiacoli JM109 strain, and the resulting Escherichia coli was applied tointernational deposition as E. coli JM109 (pNPI303) [National Instituteof Bioscience and Human Technology, Agency of Industrial Science andTechnology, the Ministry of International Trade and Industry (Higashi1-1-3, Tsukuba-shi, Ibaraki, Japan, 305), international accession numberFERM BP-5927, original deposition under Budapest Treaty on Apr. 23,1997].

II. Introduction of pNPI303 into Tobacco and Analysis ofpNPI303-introduced Tobacco

In the same manner as described in steps II and III of Example 1,plasmid pNPI303 was introduced into A. tumefaciens strain LBA 4404, aleaf disc of tobacco was infected with the resulting A. tumefaciensstrain LBA 4404 and then thus infected leaf disc was laid on thehormone-free MS agar medium containing 50 mg/l of acetosyringone andcultured for 3 days under light. Next, when the resulting infected leafwas transplanted on the hormone-free MS agar medium containing only 500mg/l of carbenicillin and the culturing was continued while subculturingwith the same medium, extreme shooty phenotypes were obtained, so that30 lines of these extreme shooty phenotypes obtained after 2 months ofthe culturing were divided into four groups and each of them was put onthe hormone-free MS agar medium containing 500 mg/l of carbenicillin, towhich 0, 10, 20 or 30 mg/l of2,2,5-trimethyl-3-(dichloroacetyl)-1,3-oxazolidine was further added, toexamine a generation ratio of normal individuals.

The results after one month of the culturing in the presence of2,2,5-trimethyl-3-(dichloroacetyl)-1,3-oxazolidine are shown in Table 2.In this case, detection of normal individuals was carried out visually,and a GUS activity test was carried out in accordance with the method ofJefferson et al. in order to confirm expression of the GUS gene used asa model of the desired gene in the example.

TABLE 2 Generation ratio of normal individuals in cultured tobaccotissue transformed with pNPI303 and concentration of 2,2,5-trimethyl-3-(dichloroacetyl)-1,3-oxazolidine The number of normal ConcentrationConcentration Concentration Concentration individuals generated 0 (mg/l)10 (mg/l) 20 (mg/l) 30 (mg/l) The number of lines*¹ 0 5 7 6 The numberof shoots*² 0 (0*³) 5 (2*³) 9 (4*³) 9 (5*³) One month after theculturing in the presence of2,2,5-trimethyl-3-(dichloroacetyl)-1,3-oxazolidine *¹The number of lineswhich generated normal individuals *²The number of shoots generated asnormal individuals *³The number of individuals among the shootsgenerated as normal individuals, which showed GUS activity

As apparent from the results shown in Table 2, no normal individual wasgenerated when no 2,2,5-trimethyl-3-(dichloroacetyl)-1,3-oxazolidine wasadded. On the other hand, normal individuals were generated when it wasadded in an amount of 10 mg/l, and the generation ratio was furtherincreased when it was added in an amount of 20 mg/l. Consequently, it isfound also in this case that the GST-II-27 promoter as a chemicalsubstance-responding promoter used in the regulation of a removable DNAelement can function properly, so that it induces expression of theremovable DNA element and accelerates its removing, and furthermoreremoving of the morphological abnormality induction gene as a selectablemarker gene, when a gene is introduced into a plant tissue by pNPI303and the resulting tissue is cultured in the presence of an appropriatechemical substance.

On the other hand, expression of the GUS activity, namely the presenceand expression of the desired gene, was confirmed in about half of theshoots generated as normal individuals, but the GUS activity was notdetectable in the remaining half. Though the reason for this is not yetclear, it is preferred, for example that, if the T-DNA region of pNPI303is introduced into the tobacco chromosome in the plural by adjoiningeach other, homologous recombination may occur not between therecombination sequence Rs's within the T-DNA region but betweenrecombination sequences which are present in mutually different T-DNAregions, thus resulting in the removing of the region sandwiched by suchsequences. As a result, not only the ipt gene but also the GUS genecould be included in the region which leaves together with the removableDNA element, and, in that case, normal individuals but having no GUSactivity would be generated from extreme shooty phenotypes similar tothe case of this example.

Also, by subjecting one of the normal individuals which showed the GUSactivity to DNA analysis by PCR, the presence of the GUS gene andremoving of the ipt gene as a selectable marker gene along with theremovable DNA element were confirmed also at the DNA level.

Industrial Applicability

When introduction of a gene into a plant cell is carried out using thevector of the present invention, a selectable marker gene introducedalong with the desired gene disappears the function thereof by removingat a certain ratio from the DNA where it exists and functions, caused bythe application of specific stimulus, such as heat, light, chemicalsubstance, or the like, to the cell after the gene introduction, and thecell into which the desired gene alone is introduced in such a mannerthat it can express on the same DNA can be obtained. Accordingly, thisvector causes the multiple introduction relating to the gene into acertain plant by merely changing the portion of the desired gene to beintroduced without any changing the structures of the selectable markergene and the others. Thus, the multiple introduction can be conducted anunlimited number of times.

Besides, since a morphological abnormality induction gene was used asthe selectable marker gene, the selection of a tissue formed solely froma cell into which the selectable marker gene was introduced, namely atissue formed solely from a cell into which the desired gene wasintroduced, as well as the selection of a tissue formed solely from acell into which the desired gene alone was introduced in such a mannerthat it can express after the disappearance of the function of theselectable marker gene, can be carried out using morphological change ofthe tissue as an index. Consequently, a tissue solely derived from acell in which the desired gene alone is introduced into chromosome orthe like can be selected surely and easily, and there is no problem ofreducing activities of the plant cell during the selection, because itis not necessary to add antibiotics for selection to the medium.Accordingly, multiple introduction of genes can be carried outefficiently, and a transgenic individual solely composed of such cells,namely an individual from which influences of the selectable marker geneare removed and anxieties about the gene product are completely cleared,can also be obtained without a crossing step.

Moreover, according to the vector of the present invention, removing ofthe selectable marker gene can be controlled artificially. Consequently,even in the case of a removable DNA element which has markedly excellentremoving ability but removes the selectable marker gene so quickly andmakes it rather difficult to obtain a tissue solely composed of cellsinto which a desired gene alone is introduced when such a regulationcannot be conducted, its ability can be used as the removable DNAelement of the present invention. On the other hand, since thegeneration of such cells, as well as the generation of plant tissuescomposed of such cells, can be optionally synchronized or controlled byusing the vector of the present invention, it becomes markedlyconvenient in actually producing transgenic plants. For example, whenthe ipt gene is used as a morphological abnormality induction gene whichis a selectable marker gene, it induces markedly active growth of a cellinto which this gene has been introduced and causes differentiation ofadventitious buds and the like under plant hormone-free conditions.Accordingly, a tissue, which can generate a cell into which a desiredgene alone is introduced at any time when it is desired, can be producedin a large scale by continuing its culturing rather under its pre-stagecondition in which the selectable marker gene is maintained.

What is claimed is:
 1. A vector suitable for introducing a gene into aplant, comprising: a desired gene, a morphological abnormality inductiongene which is capable of functioning as a selectable marker gene, and aremovable DNA element which is removed by expression of a genecatalyzing the removal, said gene being under the control of aninducible promoter, wherein the morphological abnormality induction geneis present within the removable DNA element, and wherein the desiredgene is positioned such that it is not removed together with theremovable DNA element.
 2. The vector of claim 1, wherein the induciblepromoter which controls the removable DNA element is the promoter ofribulose-bisphosphate carboxylase small subunit gene (rbcS).
 3. Thevector of claim 1, wherein the inducible promoter which controls theremovable DNA element is the promoter of glutathione-S-transferase IIsystem (GST-II) gene.
 4. The vector of claim 1, wherein the removableDNA element is derived from a site-specific recombination system.
 5. Thevector of claim 1, wherein the morphological abnormality induction geneis obtained from a bacteria belonging to the genus Agrobacterium.
 6. Thevector of claim 1, wherein the morphological abnormality induction geneis a cytokinin synthesis gene.
 7. The vector of claim 6, wherein thecytokinin synthesis gene is the ipt, isopentenyl transferase, gene whichis present in the T-DNA of Agrobacterium tumefaciens.
 8. The vector ofclaim 1, wherein the morphological abnormality induction gene induces amorphological change selected from the group consisting of abnormaldifferentiation, destruction of apical dominance, change in pigments,formation of a crown gall, formation of hairy roots and waving of theleaves.